First time using frozen yeast. Does this look healthy?

[Kornssj]

Hi All,

It's been a while and I'm glad to be back doing what I love. Coming back into the mix I was able to hit my gravity fairly better than in the past. Mostly due to my use of really thick blankets for my mash. I also mash 10 minutes longer than usually and only have to turn the heat on once during the entire 80-90 min mash. Just excited about this so i had to share!

I'm brewing an American Pale Ale (Modified soulless hop ale kit from more beer). I decided to use my harvested yeast for the first time. I created a 1L starter with 3 small vials of US-05 American and light DME. The starter was good but not vigorous as I have had in the past, most likely due to the lack of yeast cells. This had me worried about my final product once i poured into the fermenter.

I believe US-05 is notorious for slow start but I finally saw something happening and this is what it's looking like 39 hours in.

Question is, does this look healthy for that strain of yeast and this many hours in?

 

[bailey mountain brewer]

Welcome back!

Looks like fermentation is going, at just under the 2 day mark, yeah a little slow but it could have actually started before that and the activity just not visible.. what temp did you pitch and what temp is it at now? Hard to tell in the picture but I don't see anything alarming.

 

[Kornssj]

Welcome back!

Looks like fermentation is going, at just under the 2 day mark, yeah a little slow but it could have actually started before that and the activity just not visible.. what temp did you pitch and what temp is it at now? Hard to tell in the picture but I don't see anything alarming.

I brought the wort up to 66F and pitched my starter. It's been holding steady from the ink bird between 65F and 67F.

I did hear that US-05 starts off slow and around 3rd or 4th day it will take off. Seems like I'm in the clear but just wanted to check if anyone has experience with this yeast and if it looks pretty normal.

If i did slightly underpitch, can that be made up by letting the beer ferment longer?

 

[bailey mountain brewer]

Yeah, I think you're fine. And the temp you have it at is good.

 

[Kornssj]

I don't know what is going on but i just took a gravity reading at 6 days in and showing 1.042 and i started with 1.055.

Everything looks fine. I'm waiting for my sample to fully de gas but I'm thinking of gently swirling the batch to get the yeast back into suspension. Any issues with that besides krausen ending up in the beer. I assume a cold crash would clean this up at the end.

Please let me know, starting to get worried. I may have to skip re-using yeast until i get a better handle on how much i need to pitch.

 

[day_trippr]

As attenuation curves go, that's quite "flat" and strongly suggests a sub-optimal pitch.
The krausen looks thin and frankly "wimpy" for US-05 36 hours from pitching...

 

[Tony B]

Did you use a hydrometer for the gravity measurement of your recent sample?

 

[Bassman2003]

I did not catch the batch size. Also, how small were your vials of yeast coming out of frozen?

 

[Kornssj]

Did you use a hydrometer for the gravity measurement of your recent sample?

Yes just a basic wine and beer hydrometer. It does the job. I'm going to get another reading tomorrow mid day.

I did not catch the batch size. Also, how small were your vials of yeast coming out of frozen?

Batch size is 5.25 gal.

3 frozen vials see attached photo. I made a starter typical in a 2L Jug, but only had it going about 24 hours. It started going pretty well. See the other photo, that was Wednesday.

 

[mashdar]

I recommend stepping up starters if you're starting with a small or otherwise weak sample. If the starter isn't truly roaring, the cell count isn't going to be what you expect.*

General rule of thumb is 6x-10x the yeast population per step. Hard to say what you're starting with, though. Did Kai have some frozen yeast vitality stats? I feel like someone did staining/counts and wrote it up.

*edit: I regularly use larger 1yo refrigerated samples, which need 2-3 steps at least.

 

[Bassman2003]

Thanks for the info. Yes, it is important to remember that even though the yeast freezing works, there is still strain on the cells during that time in the fridge as well as a death rate. So taking samples out of the freezer requires care when you 'bring them back to life'. Meaning smaller starter steps with 1.020 wort then after signs of life and activity a step up to a larger amount of 1.040 wort.

My advice for the next time you do this:

Take the vials out of the freezer and let them sit on the counter for 24 hours.
Dump them into 250ml of 1.020 wort and let that go for 48 hours.
Take all of that and dump it into 2L of 1.040 wort.

Yes, it takes more time and wort but the yeast make the rules. It is up to us to follow them.

My guess is your current batch is not going to attenuate down to the level you are hoping for. Basically and underpitch of tired cells. The key to freezing/long term yeast storage is building up plenty of fresh new cells when you want to use them. Think of it as a worm hole that needs special attention on the other side.

 

[mac_1103]

If i did slightly underpitch

Unfortunately it seems that you massively underpitched.

Take the vials out of the freezer and let them sit on the counter for 24 hours.

Agree with everything else you wrote, but this is absolutely not the way we brought frozen cultures back to life when I used to do it for a living. Viability will decline pretty rapidly at ambient temperature if you don't get the yeast into an environment where they can start growing. You should have the media (wort) ready to inoculate when you thaw your vials, thaw them rapidly in warm water and inoculate your first starter step immediately.

 

[Tony B]

Thanks for the info. Yes, it is important to remember that even though the yeast freezing works, there is still strain on the cells during that time in the fridge as well as a death rate. So taking samples out of the freezer requires care when you 'bring them back to life'. Meaning smaller starter steps with 1.020 wort then after signs of life and activity a step up to a larger amount of 1.040 wort.

My advice for the next time you do this:

Take the vials out of the freezer and let them sit on the counter for 24 hours.
Dump them into 250ml of 1.020 wort and let that go for 48 hours.
Take all of that and dump it into 2L of 1.040 wort.

Yes, it takes more time and wort but the yeast make the rules. It is up to us to follow them.

My guess is your current batch is not going to attenuate down to the level you are hoping for. Basically and underpitch of tired cells. The key to freezing/long term yeast storage is building up plenty of fresh new cells when you want to use them. Think of it as a worm hole that needs special attention on the other side.

This is more or less the method I have moved to. I generally make 250ml of a 1.025ish starter as a first step in my 2L flask. After 48 hrs, I’ll add another 1L of 1.037 wort and let that work for at least another 48 hours.

 

[Kornssj]

Okay im going to get another starter going and repitch half a packet I bought.

Edit: Ended up doing the whole packet.

 

[Bassman2003]

Unfortunately it seems that you massively underpitched.

Agree with everything else you wrote, but this is absolutely not the way we brought frozen cultures back to life when I used to do it for a living. Viability will decline pretty rapidly at ambient temperature if you don't get the yeast into an environment where they can start growing. You should have the media (wort) ready to inoculate when you thaw your vials, thaw them rapidly in warm water and inoculate your first starter step immediately.

Thanks for your information. I freeze in 50ml vials - 50% glycerin/wort and 50% slurry. Hard pack is probably 10ml of yeast. The time when I take the vial out to the next day it seems to have some activity from the wort being in there. I assumed this was acting like a mini-starter in its (fairly) sterile environment and was giving it time to complete its task and perk up.

So with a rapid warming, how does one know when the vial is ready to pitch?

Also, why is there cell death going to or at ambient? I thought yeast were happy at ambient temps?

Always looking to learn about yeast as biology is not my forte. Thanks.

 

[Bassman2003]

This is more or less the method I have moved to. I generally make 250ml of a 1.025ish starter as a first step in my 2L flask. After 48 hrs, I’ll add another 1L of 1.037 wort and let that work for at least another 48 hours.

I would add 2L instead of 1L. The object is to grow new yeast cells and the number of cells created will be proportional to the amount of food available. One has to decide if your start is a 'vitality' starter or a 'new growth' starter. If you are trying to grow new cells it is all about the amount of wort you feed them.

 

[mac_1103]

So with a rapid warming, how does one know when the vial is ready to pitch?

As soon as the last ice crystal is gone.

Also, why is there cell death going to or at ambient? I thought yeast were happy at ambient temps?

It's not the temperature, it's the relative paucity of food and nutrients and the relatively high osmotic pressure from the glycerol.

 

[Bassman2003]

Thanks for the tip. I will change my SOP.

 

[mac_1103]

Thanks for the tip. I will change my SOP.

What you've been doing has obviously been working well for you, so it's clearly not that big a hit with your specific process. But it seems that maybe OP was starting with smaller vials and fewer cells and this could make a much bigger difference under those conditions.

 

[Bassman2003]

Well, freezing yeast has worked pretty well but I am always wanting to improve my yeast handling. I realized by adding Fermax to my pressure canned starter wort that the wort was deficient in amino acids or FAN. I do not know if this is due to the pressure canning process or my mashing is creating low-FAN wort. Starters have been much more vibrant since. The yeast performance in the ferment is ok but I think any way to get more cells viable in the early stage would help in the number of overall cells created.

The drawback of the freezing process is that you start with a low number of cells. So it is up to you to get the count up compared to if you purchased a pack of yeast and put it in a 2L starter. (my standard approach for ales). I think I underpitched at times even while trying not to due to yeast health and cell numbers. I speak mainly towards lager brewing where getting up to 400-800B cells for a 5 gallon batch is quite a bit from a vial or two. This is for cold lager fermentation and possible pressure on top of that.